FRET microscopy

The complexity of the competition between nonradiative energy transfer and charge transfer is presented in this work. This study uses ZnO nanocrystals as a donor component and rhodamine B (RhB) dye as an acceptor component. Investigations reveal that the concentration of intrinsic defects and their localization, particularly oxygen vacancies, zinc interstitials, and oxygen interstitials, play a vital role in nonradiative energy transfer from ZnO nanocrystals to RhB dye. Additionally, photoluminescent spectra indicate that ZnO nanocrystals degrade the emission signals of RhB dye via charge transfer. It is possible that a part of oxygen vacancies may also contribute to the photocatalytic oxidation of the RhB molecule. The correlation between electron paramagnetic resonance, photoluminescence, and the RhB emission decay rates indicates two processes that one may encounter when dealing with semiconductor-dye conjugates. First is a complex energy transfer from ZnO to RhB indicating a photoluminescent up-conversion and second is a charge transfer from photoexcited intrinsic defect species in ZnO suppressing the RhB emission signals, which occur together. The photoluminescent up-conversion in ZnO+RhB is due to a nonradiative energy transfer from photoexcited zinc interstitials or doubly charged oxygen vacancies in ZnO to RhB. Additionally, the suppression of RhB emission occurs due to charge transfer from oxygen vacancies in ZnO.

We have mutated a conserved residue of the death domain of the interleukin-1 (IL-1) receptor-associated kinase (IRAK), threonine 66. The substitution of Thr-66 with alanine or glutamate prevented spontaneous activation of NF-B by overexpressed IRAK but enhanced IL-1-induced activation of the factor. Like the kinaseinactivating mutation, K239S, the T66A and T66E mutations interfered with the ability of IRAK to autophosphorylate and facilitated the interactions of IRAK with TRAF6 and with the IL-1 receptor accessory protein, AcP. Wild-type IRAK constructs tagged with fluorescent proteins formed complexes that adopted a punctate distribution in the cytoplasm. The Thr-66 mutations prevented the formation of these complexes. Measurements of fluorescence resonance energy transfer among fluorescent constructs showed that the Thr-66 mutations abolished the capacity of IRAK to dimerize. In contrast, the K239S mutation did not inhibit dimerization of IRAK as evidenced by fluorescence resonance energy transfer measurements, even though microscopy showed that it prevented the formation of punctate complexes. Our results show that Thr-66 plays a crucial role in the ability of IRAK to form homodimers and that its kinase activity regulates its ability to form high molecular weight complexes. These properties in turn determine key aspects of the signaling function of IRAK. All of the biological effects of interleukin 1 (IL-1) 1 depend upon its binding to the type I IL-1 receptor, IL-1RI (1). The receptor is the prototypic member of a diverse family of conserved proteins generally involved in host defense (2). They include the Drosophila Toll protein (3), the mammalian Tolllike receptors, the IL-18 receptor and components of IL-1 signal transduction pathways such as the IL-1RI accessory protein

Flanking Domains on p300/CBP Acetyltransferase Activity 2434 5.3. Compounds That Block or Mimic p300/CBP Ligand Binding 2436 6. Potential Applications for Inhibitors and Activators of p300/CBP 2438 6.1. Loss of p300/CBP Activity in Human Disease 2438 6.2. Overexpression, Inappropriate Activation, or Mistargeting of p300/CBP 2439 6.3. Cell-Based Assays of p300/CBP Activity and Inhibition 2441 6.4. Overlap between Genetic and Pharmacologic Disruption of p300/CBP 2442 7. Future Directions 2443 8. Conclusion 2443 Associated Content 2443

DNA molecules of Vibrio cholerae and Aeromonas species were prepared by incubating immobilized cells for 4 and 2 h, respectively, with a nonenzymatic solution that contains chemical reagents only (NDSUPlus). This method gave results as reproducible as the enzymatic one that uses proteinase K, and rendered DNA molecules suitable for fingerprinting by mini-CHEF electrophoresis. As rapid DNA separations at high electric field are achieved in mini-CHEF chamber with low heat evolution, DNA restriction fragments were separated in 5 h at 10 V/cm in a single resolution window. Then, fragment separations in three resolution windows were done in 15 h. This time is shorter than the one needed by the large CHEF chamber for resolving fragments in a single resolution window. Three windows permitted to include larger numbers of restriction fragments in the calculation of isolate similarities. Both sample preparation and mini-CHEF electrophoresis may represent an alternative for performing massive epidemiological studies of V. cholerae and Aeromonas species.

We present a time-correlated single photon counting (TCPSC) technique that allows time-resolved multi-wavelength imaging in conjunction with a laser scanning microscope and a pulsed excitation source. The technique is based on a four-dimensional histogramming process that records the photon density over the time of the fluorescence decay, the x-y coordinates of the scanning area, and the wavelength. The histogramming process avoids any time gating or wavelength scanning and, therefore, yields a near-perfect counting efficiency. The time resolution is limited only by the transit time spread of the detector. The technique can be used with almost any confocal or two-photon laser scanning microscope and works at any scanning rate. We demonstrate the application to samples stained with several dyes and to CFP-YFP FRET.

In parallel to monomeric epigenetic regulators, sequence-specific epigenetic regulators represent versatile synthetic dual-target ligands that achieve regulatory control over multi-gene networks. Development of DNA-binding domain (DBD)-HDAC inhibitors and DBD-HAT activators, which result in increased histone acetylation, has become one promising research field. However, there is no report regarding the gene regulatory pattern by sequence-specific epigenetic repressor. We report here for the first time, the synthesis of DBD-HAT inhibitors and demonstrate that these conjugates could retain their dual-target activity using predicted working model of thermal stability assay and in vitro HAT activity assay. Evaluation of antiproliferative activity in cancer cells showed that 2 (with a medium linker length of 13-atom) exhibited the highest antiproliferative activity in p53 wild-type cancer cell lines (IC 50 of 1.8-2.6 µM in A549 and MV4-11 cells) and not in p53 mutant cancer cell lines. A mechanistic investigation using microarray analysis and an apoptotic assay showed that the antiproliferative effect of 2 occurred via the up-regulation of p53 target genes, and the subsequent initiation of p53-dependent apoptosis. Our research on sequence-specific dual-target epigenetic repressor offers us an alternative way to modulate HAT-governed therapeutically important genes and contributes to offer a fresh insight into antitumor therapeutics.

We propose cost-effective protocols for preparing and resolving the PulseNet universal DNA size standard in contour-clamped homogeneous electric field (CHEF) minichambers. Intact DNA molecules were prepared with protease-free solutions, and electrophoresis separations of the DNA standards needed 5.5 h, giving band pattern resolutions similar to those attained with the PulseNet protocols standardized in CHEF chambers.

The incorporation of ceramide in phase-separated monolayers of ternary lipid mixtures has been studied by a combination of atomic force microscopy (AFM), fluorescence, and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Replacement of a fraction of the sphingomyelin by ceramide in DOPC/SM/cholesterol monolayers leads to changes in the SM-cholesterol-rich liquid-ordered domains. AFM shows the formation of heterogeneous domains with small raised islands that are assigned to a ceramide-rich gel phase. ToF-SIMS provides conclusive evidence for the localization of SM and ceramide in ordered domains and shows that ceramide is heterogeneously distributed in small islands throughout the domains. The results indicate the utility of combining AFM and ToF-SIMS for understanding compositions of phase-separated membranes.

Spectral, kinetic, and nonlinear optical regularities that demonstrate the exchange of electronic excitations between the components of hybrid associates of Ag 2 S colloid quantum dots (1.7-1.8 nm) in gelatin with molecules of thiazine dyes (Ds) are found. When the IR luminescence of Ag 2 S quantum dots (QDs) is excited by radiation from the thionine absorption region, its enhancement due to nonradiative resonant energy transfer is observed. The association with methylene-blue molecules blocked the IR luminescence of Ag 2 S QDs upon its excitation by radiation from the absorption region of the dye due to the transfer of charge carriers. It is demonstrated that the hybrid association of thionine molecules and Ag 2 S QDs adversely affects the nonlinear optical properties of the latter, which manifests itself in inverse saturated absorption by the action of 10-ns second-harmonic pulses (532 nm) of a Nd 3+ :YAG laser. For the associates of Ag 2 S QDs with methylene-blue molecules, the radiation focusing caused by the transfer of charge carriers from the dye and the change in the population of small traps in nanocrystals is found. It is concluded that the direction of the transfer of electronic excitations and the photophysical processes in these objects are determined by the mutual arrangement of the HOMO-LUMO levels of the dye with respect to the levels of dimensional quantization of the Ag 2 S QDs.

Mediated through the combined action of STIM proteins and Orai channels, store-operated Ca entry (SOCE) functions ubiquitously among different cell types. The existence of multiple STIM and Orai genes has made it difficult to assign specific roles of each STIM and Orai homolog in mediating Ca signals. Using CRISPR/Cas9 gene editing tools, we generated cells with both STIM or all three Orai homologs deleted and directly monitored store Ca and Ca signals. We found that unstimulated, SOCE null KO cells still retain 50~70% of ER Ca stores of wildtype (wt) cells. After brief exposure to store-emptying conditions, acute refilling of ER Ca stores was totally blocked in KO cells. However, after 24 h in culture, stores were eventually refilled. Thus, SOCE is critical for immediate refilling of ER Ca but is dispensable for the maintenance of long-term ER Ca homeostasis. Using the Orai null background triple Orai-KO cells, we examined the plasma membrane translocation properties of a series of...

We report the formation and photonic properties of CdSe/ZnS quantum dot (QD)/J-aggregate donor-acceptor Forster resonant energy transfer (FRET) pairs that are electrostatically bound and dispersed in water. Assembly occurs when the positively charged dye, 1,1′,3,3′-tetraethyl-5,5′,6,6′-tetrachlorobenzimidazolocarbocyanine (TTBC), binds electrostatically to QDs that are coated with a negatively charged amphiphilic polymer. QD/ J-aggregate FRET pairs display the broadband absorption in the visible and the ultraviolet (UV) part of the spectrum typical of quantum dots, along with the narrow emission linewidths characteristic of J-band emitters (∼12 nm full width at half-maximum). We use dynamic light scattering, transmission electron microscopy, photoluminescence spectroscopy, and photoluminescence lifetimes to conclude that the size of the aggregates formed in the presence of QDs is between 1-6 nm. We find the FRET radius of the QD/J-aggregate to be 5.1 nm. We further demonstrate electrostatic binding as a general synthetic strategy for these types of FRET pairs by attaching a negatively charged J-aggregate (BIC) to a quantum dot coated with positively charged ligands.

We present a time-correlated single photon counting (TCPSC) technique that allows time-resolved multi-wavelength imaging in conjunction with a laser scanning microscope and a pulsed excitation source. The technique is based on a four-dimensional histogramming process that records the photon density over the time of the fluorescence decay, the x-y coordinates of the scanning area, and the wavelength. The histogramming process avoids any time gating or wavelength scanning and, therefore, yields a near-perfect counting efficiency. The time resolution is limited only by the transit time spread of the detector. The technique can be used with almost any confocal or two-photon laser scanning microscope and works at any scanning rate. We demonstrate the application to samples stained with several dyes and to CFP-YFP FRET.

Store-operated Ca 2 þ entry mediated by STIM1 and ORAI1 constitutes one of the major Ca 2 þ entry routes in mammalian cells. The molecular choreography of STIM1-ORAI1 coupling is initiated by endoplasmic reticulum (ER) Ca 2 þ store depletion with subsequent oligomerization of the STIM1 ER-luminal domain, followed by its redistribution towards the plasma membrane to gate ORAI1 channels. The mechanistic underpinnings of this inside-out Ca 2 þ signalling were largely undefined. By taking advantage of a unique gain-of-function mutation within the STIM1 transmembrane domain (STIM1-TM), here we show that local rearrangement, rather than alteration in the oligomeric state of STIM1-TM, prompts conformational changes in the cytosolic juxtamembrane coiled-coil region. Importantly, we further identify critical residues within the cytoplasmic domain of STIM1 (STIM1-CT) that entail autoinhibition. On the basis of these findings, we propose a model in which STIM1-TM reorganization switches STIM1-CT into an extended conformation, thereby projecting the ORAI-activating domain to gate ORAI1 channels.

We propose cost-effective protocols for preparing and resolving the PulseNet universal DNA size standard in contour-clamped homogeneous electric field (CHEF) minichambers. Intact DNA molecules were prepared with protease-free solutions, and electrophoresis separations of the DNA standards needed 5.5 h, giving band pattern resolutions similar to those attained with the PulseNet protocols standardized in CHEF chambers.

We present a time-correlated single photon counting (TCPSC) technique that allows time-resolved multi-wavelength imaging in conjunction with a laser scanning microscope and a pulsed excitation source. The technique is based on a four-dimensional histogramming process that records the photon density over the time of the fluorescence decay, the x-y coordinates of the scanning area, and the wavelength. The histogramming process avoids any time gating or wavelength scanning and, therefore, yields a near-perfect counting efficiency. The time resolution is limited only by the transit time spread of the detector. The technique can be used with almost any confocal or two-photon laser scanning microscope and works at any scanning rate. We demonstrate the application to samples stained with several dyes and to CFP-YFP FRET.

We present a fluorescence lifetime imaging technique with simultaneous spectral and temporal resolution. The technique is fully compatible with the commonly used multiphoton microscopes and nondescanned (direct) detection. An image of the back-aperture of the microscope lens is projected on the input of a fiber bundle. The input of the fiber bundle is circular, and the output is flattened to match the input slit of a spectrograph. The spectrum at the output of the spectrograph is projected on a 16-anode PMT module. For each detected photon, the encoding logics of the PMT module deliver a timing pulse and the number of the PMT channel in which the photon was detected. The photons are accumulated by a multidimensional time-correlated single photon counting (TCSPC) process. The recording process builds up a four-dimensional photon distribution over the times of the photons in the excitation pulse period, the wavelengths of the photons, and the coordinates of the scan area. The method delivers a near-ideal counting efficiency and is capable of resolving double-exponential decay functions. We demonstrate the performance of the technique for autofluorescence imaging of tissue.

When and where proteins associate with each other in living cells are key questions in many biological research projects. One way to address these questions is to measure the extent of Förster resonance energy transfer (FRET) between proteins that have been labeled with appropriate donor and acceptor fluorophores. When both proteins interact, donor and acceptor fluorophores are brought into close vicinity so that the donor can transmit a part of its excitation energy to the acceptor. As a result, both the intensity and the lifetime of the donor fluorescence decrease, whereas the intensity of the acceptor emission increases. This offers different approaches to determine FRET efficiency: One is to detect changes in the intensity of donor and acceptor emission, the other is to measure changes in the lifetime of the donor molecule. One important advantage of the fluorescence lifetime approach is that it allows to distinguish between free and associated donor molecules. However, like intensity measurements it lacks an intrinsic control ensuring that changes in the measured parameters are only due to FRET and not other quenching processes. Here, we show how this limitation can be overcome by spectrally resolved fluorescence lifetime measurements in the time domain. One technique is based on a streak camera system, the other technique is based on a time-correlated-single-photon-counting approach. Both approaches allow biologists to record both donor and acceptor fluorescence emitted by the sample in a single measurement.

These pages are an attempt to help existing and future users of the bh FLIM technique obtain the best possible results from their FLIM experiments. The first part of the brochure explains the principle of TCSPC FLIM, and gives an impression of the photon distributions recorded. It shows that the signal-to-noise ratio of the measured lifetimes depends, in first order, on the number of photons recorded.  The following sections focus on optimising the photon number without increasing the photostress imposed to the sample. We discuss the influence of excitation power, acquisition time, collection efficiency, numerical aperture, focusing precision, alignment accuracy, and detector efficiency. The next section concentrates on photon efficiency. It considers TCSPC timing parameters, counting background, number of pixels, the influence of the instrument-response function, and the challenges of multi-exponential decay functions. The last section is dedicated to data analysis. All conclusions made in this brochure are demonstrated on real measurement data recorded under realistic conditions.

CBP and EP300 are highly homologous, bromodomain-containing transcription coactivators involved in numerous cellular pathways relevant to oncology. As part of our effort to explore the potential therapeutic implications of selectively targeting bromodomains, we set out to identify a CBP/EP300 bromodomain inhibitor that was potent both in vitro and in cellular target engagement assays and was selective over the other members of the bromodomain family. Reported here is a series of cell-potent and selective probes of the CBP/EP300 bromodomains, derived from the fragment screening hit 4-methyl-1,3,4,5-tetrahydro-2H-benzo[b][1,4]diazepin-2-one.

Spectral, kinetic, and nonlinear optical regularities that demonstrate the exchange of electronic excitations between the components of hybrid associates of Ag 2 S colloid quantum dots (1.7-1.8 nm) in gelatin with molecules of thiazine dyes (Ds) are found. When the IR luminescence of Ag 2 S quantum dots (QDs) is excited by radiation from the thionine absorption region, its enhancement due to nonradiative resonant energy transfer is observed. The association with methylene-blue molecules blocked the IR luminescence of Ag 2 S QDs upon its excitation by radiation from the absorption region of the dye due to the transfer of charge carriers. It is demonstrated that the hybrid association of thionine molecules and Ag 2 S QDs adversely affects the nonlinear optical properties of the latter, which manifests itself in inverse saturated absorption by the action of 10-ns second-harmonic pulses (532 nm) of a Nd 3+ :YAG laser. For the associates of Ag 2 S QDs with methylene-blue molecules, the radiation focusing caused by the transfer of charge carriers from the dye and the change in the population of small traps in nanocrystals is found. It is concluded that the direction of the transfer of electronic excitations and the photophysical processes in these objects are determined by the mutual arrangement of the HOMO-LUMO levels of the dye with respect to the levels of dimensional quantization of the Ag 2 S QDs.

Amphiphilic polymers have emerged as an important class of materials owing to their ability to produce a diverse range of self-assembled structures with container properties that can be used to address growing challenges in biomedical applications. Thus, in-depth understanding on their aggregation properties is important fundamentally as well as from an application viewpoint. In this article we describe utilization of fluorescence resonance energy transfer (FRET) as a powerful tool to elucidate various physical properties of amphiphilic block copolymer aggregates at very low polymer concentration (∼10 -7 M) which otherwise are difficult to achieve using other commonly used techniques such as microscopy, scattering, or external probe based spectroscopic techniques. We synthesized a prepolymer based on PEO-b-PMMA-co-PHEMA and subsequently utilized the hydroxy groups of the HEMA units to covalently attach with either a green (D) or a red (A) fluorescent dye (D-A pair suitable for FRET) to get D-and A-labeled amphiphilic polymers with similar spatial distribution of these chromophores in the hydrophobic block of the two polymers. Coassembly of red and green labeled polymers in micellar aggregates results in highly efficient FRET while no FRET is observed when they remain as unimer. This was exploited to study the micellization process by solvent, concentration, time, and pH dependent FRET studies either by mixing preformed aggregates of the two polymers or by inducing aggregation in mixture of unimers. Contrary to existing perception, our studies revealed exceptionally slow dynamics (mixing time >50 h), very low critical aggregation concentration (<10 -7 M), remarkably high tolerance to good solvent, and intriguing solvent induced swelling followed by disassembly of the micellar aggregate from this rather simple diblock copolymer. We explicitly show why the FRET based tool stands out among other techniques to probe such detail physical characteristics of micellar aggregates with precision at very dilute concentration which otherwise appears to be a rather difficult task. More interestingly, we provide the rationale behind the exceptionally high stability of these micelles by providing evidence of noncovalent core cross-linking by H-bonding among the few unreacted OH groups present in the hydrophobic block. When this parameter was lifted off by either protonation at acidic pH or protecting the hydroxyl group by acetyl group, FRET studies showed very fast dynamics of the micellar aggregations, confirming the free OH groups in the hydrophobic domain are responsible for noncovalent core cross-linking leading to the unusual stability of the micelles.

The enzymes which are responsible for catalyzing sequential reactions in several metabolic pathways have been proposed to be highly organized in supramolecular complexes termed metabolons. However, the in situ existence of these weak complexes is difficult to demonstrate because many of them are dissociated during isolation due to dilution effects. Consequently, the metabolon concept is subject to controversy. A model system consisting of genetically prepared bienzymatic fusion proteins has been used to immobilize sequential metabolic enzymes in close proximity and to demonstrate possible kinetic advantages of metabolons. These experiments use the sequential Krebs TCA cycle enzymes from yeast mitochondrial malate dehydrogenase (MDH), citrate synthase (CS), and aconitase (ACO). Using the porcine high-definition structures of these three enzymes, we have performed computer-modeling studies in order to understand how the molecules may interact. Among the thousands of docking orientations we have tried, one was found to respond to the structural and experimental constraints from the results obtained with the yeast fusion proteins. Interestingly, this quinary structure model shows substantial interacting surface areas with spatial and electrostatic complementarities which make the complex thermodynamically stable. This structure also contains an unbroken electrostatically favorable channel connecting the active sites of ACO and CS, as well as the one previously reported between CS and MDH active sites. Charged amino acids which could be involved in interactions stabilizing the complex have been identified. This model will be used as the basis for further experimental work on the structure of the Krebs TCA cycle metabolon.

Three different particle sizes from cadmium telluride semiconductor quantum dots (CdTe QDs) have been prepared using thioglycolic acid as a capping agent in aqueous media. The photophysical behavior for these particles has been studied in presence of monomethine dyes belong to thiazole orange (TO) family. The aggregation of the cyanine dyes on QDs surface was attributed to the presence of electrostatic attraction between negatively charged thioglycolic capping molecules and the ionic dye. Energy transfer process from CdTe QDs to cyanine dyes was also studied using electronic absorption and steady state emission measurements. The energy transfer parameters such as Stern-Volmer rate constants Ksv, binding sites n, quenching sphere radius "r", critical energy transfer distance "R0" and the efficiency of energy transfer E, have been calculated. The enhancement in the triplet state emission and absorption for the studied dye in the presence of QDs was expected to play a role in molecular oxygen sensitization and photodynamic therapy (PDT) as well as photovoltaic applications..

Store-operated Ca 2 þ entry mediated by STIM1 and ORAI1 constitutes one of the major Ca 2 þ entry routes in mammalian cells. The molecular choreography of STIM1-ORAI1 coupling is initiated by endoplasmic reticulum (ER) Ca 2 þ store depletion with subsequent oligomerization of the STIM1 ER-luminal domain, followed by its redistribution towards the plasma membrane to gate ORAI1 channels. The mechanistic underpinnings of this inside-out Ca 2 þ signalling were largely undefined. By taking advantage of a unique gain-of-function mutation within the STIM1 transmembrane domain (STIM1-TM), here we show that local rearrangement, rather than alteration in the oligomeric state of STIM1-TM, prompts conformational changes in the cytosolic juxtamembrane coiled-coil region. Importantly, we further identify critical residues within the cytoplasmic domain of STIM1 (STIM1-CT) that entail autoinhibition. On the basis of these findings, we propose a model in which STIM1-TM reorganization switches STIM1-CT into an extended conformation, thereby projecting the ORAI-activating domain to gate ORAI1 channels.

Lactate/lactic acid has been considered as a biofuel for enzymatic biofuel cells, but only with single enzyme bioanodes containing lactate dehydrogenase. A single enzyme-based bioanode results in the oxidation of lactate to pyruvate, which only allows for 2 of the total of 12 electrons to be harnessed from the lactate leaving the majority of the energy density of the fuel in the oxidized product (pyruvate); thereby, resulting in low energy density biofuel cells. This paper details an enzyme cascade for complete oxidation of lactate immobilized on a bioanode and employed in a lactate/air biofuel cell. This paper shows that complete oxidation of lactate increases the current density and power density of the biofuel cell, in a similar trend as was observed for complete oxidation of pyruvate in an enzymatic biofuel cell.

Xia Zhang(1), Cristian A. Marocico(1), Manuela Lunz(1)†, Valerie A. Gerard (2), Yurii K. Gun’ko (2), Vladimir Lesnyak (3), Nikolai Gaponik (4), Andrei S. Susha (5), Andrey L. Rogach (5), and A. Louise Bradley (1)* (1) School of Physics and CRANN, Trinity College, Dublin 2, Ireland (2) School of Chemistry and CRANN, Trinity College, Dublin 2, Ireland (3) Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova, Italy (4) Physical Chemistry, TU Dresden, Bergstrasse 66b, 01062 Dresden, Germany (5) Department of Physics and Materials Science & Center for Functional Photonics (CFP), City University of Hong Kong, Hong Kong, China

The incorporation of ceramide in phase-separated monolayers of ternary lipid mixtures has been studied by a combination of atomic force microscopy (AFM), fluorescence, and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Replacement of a fraction of the sphingomyelin by ceramide in DOPC/SM/cholesterol monolayers leads to changes in the SM-cholesterol-rich liquid-ordered domains. AFM shows the formation of heterogeneous domains with small raised islands that are assigned to a ceramide-rich gel phase. ToF-SIMS provides conclusive evidence for the localization of SM and ceramide in ordered domains and shows that ceramide is heterogeneously distributed in small islands throughout the domains. The results indicate the utility of combining AFM and ToF-SIMS for understanding compositions of phase-separated membranes.

Förster resonance energy transfer ͑FRET͒ between CdTe quantum dots ͑QDs͒ at nanoscale proximity to gold nanoparticle ͑Au NP͒ layers is investigated experimentally. We have observed the enhancement in the acceptor QDs' photoluminescence lifetime intensities. The decrease in donor QDs' exciton lifetime from 5.74 to 2.06 ns, accompanied by an increase in acceptor QDs' exciton lifetime from 3.38 to 7.52 ns, provided evidence for enhanced FRET between the QDs near Au NPs. The Au NPs' surface plasmon dipole fields are assisted to overcome the weak electronic coupling between the emitting ͑donor͒ and absorbing ͑acceptor͒ transition exciton dipoles in the homogeneous medium.

Förster resonance energy transfer ͑FRET͒ between CdTe quantum dots ͑QDs͒ at nanoscale proximity to gold nanoparticle ͑Au NP͒ layers is investigated experimentally. We have observed the enhancement in the acceptor QDs' photoluminescence lifetime intensities. The decrease in donor QDs' exciton lifetime from 5.74 to 2.06 ns, accompanied by an increase in acceptor QDs' exciton lifetime from 3.38 to 7.52 ns, provided evidence for enhanced FRET between the QDs near Au NPs. The Au NPs' surface plasmon dipole fields are assisted to overcome the weak electronic coupling between the emitting ͑donor͒ and absorbing ͑acceptor͒ transition exciton dipoles in the homogeneous medium.

In recent years it has become clear that a cell cannot be visualized as a 'bag' filled with enzymes dissolved in bulk water. The aqueous-phase properties in the interior of a cell are, indeed, essentially different from those of an ordinary aqueous solution. Large amounts of water are believed to be organized in layers at the surface of intracellular structural proteins and membranes. Such considerations prompt us to reconsider the operation and regulation of metabolic pathways. Enzymes of metabolic pathways are nowadays thought to be clustered and operate as 'metabolons'. Very often interactions between enzymes of a pathway can exclusively be evidenced in uitro in media which are known to reduce the water concentration in the vicinity of the proteins. Immobilized enzyme preparations have been shown to be excellent tools for this type of research. We describe here some recent studies where immobilized enzymes have been used in various applications to investigate associations among enzymes of a number of different metabolic pathways (glycolysis/gluconeogenesis, citric acid cycle and its connection to the electron transport chain, aspartate-malate shuttle, glyoxylate cycle). Advantages and disadvantages of the different techniques are also discussed.

We have mutated a conserved residue of the death domain of the interleukin-1 (IL-1) receptor-associated kinase (IRAK), threonine 66. The substitution of Thr-66 with alanine or glutamate prevented spontaneous activation of NF-B by overexpressed IRAK but enhanced IL-1-induced activation of the factor. Like the kinaseinactivating mutation, K239S, the T66A and T66E mutations interfered with the ability of IRAK to autophosphorylate and facilitated the interactions of IRAK with TRAF6 and with the IL-1 receptor accessory protein, AcP. Wild-type IRAK constructs tagged with fluorescent proteins formed complexes that adopted a punctate distribution in the cytoplasm. The Thr-66 mutations prevented the formation of these complexes. Measurements of fluorescence resonance energy transfer among fluorescent constructs showed that the Thr-66 mutations abolished the capacity of IRAK to dimerize. In contrast, the K239S mutation did not inhibit dimerization of IRAK as evidenced by fluorescence resonance energy transfer measurements, even though microscopy showed that it prevented the formation of punctate complexes. Our results show that Thr-66 plays a crucial role in the ability of IRAK to form homodimers and that its kinase activity regulates its ability to form high molecular weight complexes. These properties in turn determine key aspects of the signaling function of IRAK. All of the biological effects of interleukin 1 (IL-1) 1 depend upon its binding to the type I IL-1 receptor, IL-1RI (1). The receptor is the prototypic member of a diverse family of conserved proteins generally involved in host defense (2). They include the Drosophila Toll protein (3), the mammalian Tolllike receptors, the IL-18 receptor and components of IL-1 signal transduction pathways such as the IL-1RI accessory protein

The development of direct multicolour fl uorescence cross-correlation spectroscopy: Towards a new tool for tracking complex biomolecular events in real-time HOT ARTICLE Gong et al. Superior reactivity of skeletal Ni-based catalysts for low-temperature steam reforming to produce CO-free hydrogen

The incorporation of ceramide in phase-separated monolayers of ternary lipid mixtures has been studied by a combination of atomic force microscopy (AFM), fluorescence, and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Replacement of a fraction of the sphingomyelin by ceramide in DOPC/SM/cholesterol monolayers leads to changes in the SM-cholesterol-rich liquid-ordered domains. AFM shows the formation of heterogeneous domains with small raised islands that are assigned to a ceramide-rich gel phase. ToF-SIMS provides conclusive evidence for the localization of SM and ceramide in ordered domains and shows that ceramide is heterogeneously distributed in small islands throughout the domains. The results indicate the utility of combining AFM and ToF-SIMS for understanding compositions of phase-separated membranes.

In recent years it has become clear that a cell cannot be visualized as a 'bag' filled with enzymes dissolved in bulk water. The aqueous-phase properties in the interior of a cell are, indeed, essentially different from those of an ordinary aqueous solution. Large amounts of water are believed to be organized in layers at the surface of intracellular structural proteins and membranes. Such considerations prompt us to reconsider the operation and regulation of metabolic pathways. Enzymes of metabolic pathways are nowadays thought to be clustered and operate as 'metabolons'. Very often interactions between enzymes of a pathway can exclusively be evidenced in uitro in media which are known to reduce the water concentration in the vicinity of the proteins. Immobilized enzyme preparations have been shown to be excellent tools for this type of research. We describe here some recent studies where immobilized enzymes have been used in various applications to investigate associations among enzymes of a number of different metabolic pathways (glycolysis/gluconeogenesis, citric acid cycle and its connection to the electron transport chain, aspartate-malate shuttle, glyoxylate cycle). Advantages and disadvantages of the different techniques are also discussed.

The PulseNet National Database, established by the Centers for Disease Control and Prevention in 1996, consists of pulsed-field gel electrophoresis (PFGE) patterns obtained from isolates of food-borne pathogens (currently Escherichia coli O157:H7, Salmonella, Shigella, and Listeria) and textual information about the isolates. Electronic images and accompanying text are submitted from over 60 U.S. public health and food regulatory agency laboratories. The PFGE patterns are generated according to highly standardized PFGE protocols. Normalization and accurate comparison of gel images require the use of a well-characterized size standard in at least three lanes of each gel. Originally, a well-characterized strain of each organism was chosen as the reference standard for that particular database. The increasing number of databases, difficulty in identifying an organism-specific standard for each database, the increased range of band sizes generated by the use of additional restriction endonucleases, and the maintenance of many different organism-specific strains encouraged us to search for a more versatile and universal DNA size marker. A Salmonella serotype Braenderup strain (H9812) was chosen as the universal size standard. This strain was subjected to rigorous testing in our laboratories to ensure that it met the desired criteria, including coverage of a wide range of DNA fragment sizes, even distribution of bands, and stability of the PFGE pattern. The strategy used to convert and compare data generated by the new and old reference standards is described.

Förster resonance energy transfer ͑FRET͒ between CdTe quantum dots ͑QDs͒ at nanoscale proximity to gold nanoparticle ͑Au NP͒ layers is investigated experimentally. We have observed the enhancement in the acceptor QDs' photoluminescence lifetime intensities. The decrease in donor QDs' exciton lifetime from 5.74 to 2.06 ns, accompanied by an increase in acceptor QDs' exciton lifetime from 3.38 to 7.52 ns, provided evidence for enhanced FRET between the QDs near Au NPs. The Au NPs' surface plasmon dipole fields are assisted to overcome the weak electronic coupling between the emitting ͑donor͒ and absorbing ͑acceptor͒ transition exciton dipoles in the homogeneous medium.

Sensitive detection of nucleic acids and identification of single nucleotide polymorphism (SNP) is crucial in diagnosis of genetic diseases. Many strategies have been developed for detection and analysis of DNA, including fluorescence, electrical, optical, and mechanical methods. Recent advances in fluorescence resonance energy transfer (FRET)-based sensing have provided a new avenue for sensitive and quantitative detection of various types of biomolecules in simple, rapid, and recyclable platforms. Here, we report single-step FRET-based DNA sensors designed to work via a toehold-mediated strand displacement (TMSD) process, leading to a distinct change in the FRET efficiency upon target binding. Using single-molecule FRET (smFRET), we show that these sensors can be regenerated in situ, and they allow detection of femtomoles DNA without the need for target amplification while still using a dramatically small sample size (fewer than three orders of magnitude compared to the typical sample size of bulk fluorescence). In addition, these single-molecule sensors exhibit a dynamic range of approximately two orders of magnitude. Using one of the sensors, we demonstrate that the single-base mismatch sequence can be discriminated from a fully matched DNA target, showing a high specificity of the method. These sensors with simple and recyclable design, sensitive detection of DNA, and the ability to discriminate single-base mismatch sequences may find applications in quantitative analysis of nucleic acid biomarkers.

The inverse correlation between the exciton-lattice interaction strength and the exciton delocalization length is shown for dielectric organic nanoclusters – J-aggregates. Since the exciton delocalization length defines all optical properties of J-aggregates, it allows one to control them via the exciton-lattice interaction. We demonstrate the exciton self-trapping suppression in amphi-PIC J-aggregates as a result of the exciton-lattice interaction weakening due to the formation of a surfactant shell around J-aggregates. The control over optical properties via the exciton-lattice interaction for other types of J-aggregates is discussed.

Confocal luminescence microscopy has been used to excite and collect luminescence from single amphi-PIC J-aggregate. Two types of J-aggregates have been revealed in the luminescence image: bead-like J-aggregates, which diameter is less than 1 mm and rod-like ones, which length is about 3 mm and diameter is less than 1 mm. It has been found that single rod-like and bead-like J-aggregates exhibit different luminescence bands with different decay parameters. At the off-resonance blue tail excitation, the J-aggregate exciton luminescence disappeared within a certain time period and a new band appeared, which cannot be attributed to the monomer emission. The luminescence image shows that the J-aggregate is not destroyed. However, J-aggregate storage in darkness does not recover its exciton luminescence.

The incorporation of ceramide in phase-separated monolayers of ternary lipid mixtures has been studied by a combination of atomic force microscopy (AFM), fluorescence, and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Replacement of a fraction of the sphingomyelin by ceramide in DOPC/SM/cholesterol monolayers leads to changes in the SM-cholesterol-rich liquid-ordered domains. AFM shows the formation of heterogeneous domains with small raised islands that are assigned to a ceramide-rich gel phase. ToF-SIMS provides conclusive evidence for the localization of SM and ceramide in ordered domains and shows that ceramide is heterogeneously distributed in small islands throughout the domains. The results indicate the utility of combining AFM and ToF-SIMS for understanding compositions of phase-separated membranes.

By analyzing DNA migrations per pulse at different pulse times we determined the reorientation times and migration velocities, during and after DNA reorientation of molecules separated under different clamped homogeneous electric field (CHEF) electrophoretic conditions. We obtained functions relating these parameters and DNA migration per pulse with running variables and DNA size. A statistical procedure that validates the predictions of these functions was also proposed using identical data for function fitness and validation. A method is proposed to estimate DNA size, reorientation time and migration velocities using the distance migrated by a DNA molecule in a single CHEF experiment. 0 1998 Elsevier Science B.V.